【摘要】：【目的】研究臍帶血間充質干細胞體外分離、培養(yǎng)條件，探討臍帶血間充質干細胞作為細胞治療種子細胞的可行性。 【方法】無菌條件下取正常足月產(chǎn)臍帶血36份，CAPD-1復合抗凝劑抗凝。其中18份臍血采用直接分離法，即將臍血稀釋后，直接用Ficoll分離單個核細胞，分別采用含5％、10％、20％血清濃度的L-DMEM常規(guī)間充質干細胞貼壁培養(yǎng)；其余18份臍血采用間接分離法，即將臍血用5g／L的甲基纖維素先沉降出紅細胞，然后將富集到的白細胞用Ficoll進行密度梯度離心，獲得單個核細胞，同樣分別采用含5％、10％、20％血清濃度的L-DMEM進行培養(yǎng)；另外，無菌條件下取11份骨髓，按常規(guī)間充質干細胞分離培養(yǎng)法，分離培養(yǎng)骨髓作為陽性對照。對不同方法富集到的臍血單個核細胞數(shù)量進行對比，對臍血原代和傳代細胞的貼壁時間、貼壁細胞類型、生長速度進行觀察記錄，對各臍血組成功培養(yǎng)出間充質干細胞的例數(shù)進行統(tǒng)計分析，，對分離出的臍血間充質干細胞進行鑒定并與骨髓間充質干細胞對比，采用流式細胞儀考察臍血及骨髓中單個核細胞的細胞亞群以及間充質干細胞存在情況。
[Abstract]:[objective] to study the isolation and culture conditions of umbilical cord blood mesenchymal stem cells in vitro, and to explore the feasibility of using umbilical cord blood mesenchymal stem cells as cells to treat seed cells. [methods] 36 cases of normal term cord blood were collected under aseptic condition. CAPD-1 combined with anticoagulant was used. 18 cord blood samples were separated by direct separation method. Mononuclear cells were separated directly by Ficoll after dilution of cord blood. The normal mesenchymal stem cells (MSCs) were cultured with L-DMEM containing 5% 10% serum concentration and 20% serum concentration respectively. The other 18 cord blood samples were separated indirectly, that is, the cord blood was precipitated out of red blood cells with methyl cellulose from 5g/L, and then the enriched white blood cells were centrifuged by density gradient centrifugation with Ficoll to obtain mononuclear cells. L-DMEM with 20% serum concentration was cultured. In addition, 11 samples of bone marrow were isolated and cultured as positive control according to the routine method of mesenchymal stem cell isolation and culture under aseptic condition. The number of cord blood mononuclear cells enriched by different methods was compared. The adherent time, adherent cell type and growth rate of umbilical cord blood primary and passage cells were observed and recorded. The cases of mesenchymal stem cells cultured successfully in each cord blood group were statistically analyzed. The isolated mesenchymal stem cells were identified and compared with bone marrow mesenchymal stem cells. The subsets of mononuclear cells and mesenchymal stem cells in cord blood and bone marrow were investigated by flow cytometry.
【學位授予單位】：四川大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R329

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